NFIB/MLL1 complex is required for the stemness and the Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE227538 | GEO | 2023/09/05
REPOSITORIES: GEO
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Project description:The coordinated and sequential actions of lineage-specific transcription factors and epigenetic regulators are essential for the initiation and maintenance of cellular differentiation. We here report KDM4D histone demethylase as a key regulator of adipogenesis in C3H10T1/2 mesenchymal stem cells. The depletion of KDM4D results in impaired differentiation, which can be rescued by exogenous KDM4D, PPAR?, and C/EBP?, but not by C/EBP?. In addition, KDM4D interacts physically and functionally with both NFIB and MLL1 complex to regulate C/EBP? and PPAR? expression upon adipogenic hormonal induction. Although KDM4D is dispensable for the binding of both NFIB and MLL1 complex to the target promoters, the demethylation of tri-methylated H3K9 by KDM4D is required for NFIB and MLL1 complex to deposit tri-methylated H3K4 and activate PPAR? and C/EBP? expression. Taken together, our data provide a molecular framework for lineage-specific transcription factor and histone modifiers to cooperate in adipogenic differentiation, in which KDM4D removes repressive histone marks at genes with a bivalent chromatin domain and allows NFIB and MLL1 complex to promote the expression of key adipogenic regulators.
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Project description:To explore direct targets of NFIB/MLL1 complex and to examine the role of NFIB/MLL1 complex in genome-wide deposition of histone H3-K4 tri-methylation (H3K4me3), we conducted chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analysis of six different samples at two time points.
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Project description:To examine the impact of the NFIB/MLL1 complex on the transcriptome of C3H10T1/2 mesenchymal stem cells (MSCs) and to identify NFIB/MLL1-dependent genes during osteogenic differentiation, we performed RNA-sequencing (RNA-seq) analysis of six different samples at two time points.
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Project description:To investigate the cooperative function KDM4D/NFIB/MLL1 complex in the regulation of adipogenic differentiation, we established 10T1/2 cell lines in which each target gene has been knocked down by shRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.
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Project description:Wnt/?-catenin signaling is crucial for intestinal carcinogenesis and the maintenance of intestinal cancer stem cells. Here we identify the histone methyltransferase Mll1 as a regulator of Wnt-driven intestinal cancer. Mll1 is highly expressed in Lgr5+ stem cells and human colon carcinomas with increased nuclear ?-catenin. High levels of MLL1 are associated with poor survival of colon cancer patients. The genetic ablation of Mll1 in mice prevents Wnt/?-catenin-driven adenoma formation from Lgr5+ intestinal stem cells. Ablation of Mll1 decreases the self-renewal of human colon cancer spheres and halts tumor growth of xenografts. Mll1 controls the expression of stem cell genes including the Wnt/?-catenin target gene Lgr5. Upon the loss of Mll1, histone methylation at the stem cell promoters switches from activating H3K4 tri-methylation to repressive H3K27 tri-methylation, indicating that Mll1 sustains stem cell gene expression by antagonizing gene silencing through polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation. Transcriptome profiling of Wnt-mutated intestinal tumor-initiating cells reveals that Mll1 regulates Gata4/6 transcription factors, known to sustain cancer stemness and to control goblet cell differentiation. Our results demonstrate that Mll1 is an essential epigenetic regulator of Wnt/?-catenin-induced intestinal tumorigenesis and cancer stemness.
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Project description:Bone marrow derived human mesenchymal stem cells (MSC) are a great source in bone tissue engineering. However, how to improve the efficiency of MSC osteogenesis remains a big challenge in bone regenerative medicine. Here, we characterized the role of INO80 chromatin remodeling complex in osteogenic differentiation of MSC. We showed that silencing of subunits of INO80 reduced the mineral deposition of MSC in osteogenic condition. Moreover, INO80-silencing MSC cultured in osteogenic condition expressed lower mRNA levels of osteoblast-specific genes, including Runx2, Osx, Col1?1 and OCN. INO80 can interact with Wdr5 in MSC and positively regulates the canonical Wnt signaling transduction. Importantly, the mice implanted with INO80-silencing MSC displayed less bone formation. Overall, our study provides a new mechanism regarding osteogenic differentiation of MSC and could potentially be applied in clinical tissue engineering and treatment of osteoporosis.
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Project description:The differentiation potential of mesenchymal stem cells (MSC) has been extensively tested on electrospun scaffolds. However, this potential is often assessed with lineage-specific medium, making it difficult to interpret the real contribution of the properties of the scaffold in the cell response. In this study, we analyzed the ability of different polycaprolactone/polylactic acid PCL/PLA electrospun scaffolds (pure or blended compositions, random or aligned fibers, various fiber diameters) to drive MSC towards bone or tendon lineages in the absence of specific differentiation medium. C3H10T1/2 cells (a mesenchymal stem cell model) were cultured on scaffolds for 96 h without differentiation factors. We performed a cross-analysis of the cell-scaffold interactions (spreading, organization, and specific gene expression) with mechanical (elasticity), morphological (porosity, fibers diameter and orientation) and surface (wettability) characterizations of the electrospun fibers. We concluded that (1) osteogenic differentiation can be initiated on pure PCL-based electrospun scaffolds without specific culture conditions; (2) fiber alignment modified cell organization in the short term and (3) PLA added to PCL with an increased fiber diameter encouraged the stem cells towards the tendon lineage without additional tenogenic factors. In summary, the differentiation potential of stem cells on adapted electrospun fibers could be achieved in factor-free medium, making possible future applications in clinically relevant situations.
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Project description:Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.
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Project description:Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n?=?3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5?ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB?+?FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5?ng/ml FGF-2 [ACK(AB?+?FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5?ng/ml FGF2 [Lympho(AB?+?FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL)?+?heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB?+?FGFlow) or 10% AB +10?ng/ml FGF2 [Direct(AB?+?FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB?+?FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB?+?FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB?+?FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1? and IL-1? expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB?+?FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.
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